1. A B
12T 42 51 55 60 86 92
Supplementary Figure 1: Characterization of the identified HLA peptides. (A) Logo of all HLA-I peptides of 8-13 amino acids in length identified from each patient. (B) The length distributions of the HLA-I and HLA-II peptide repertoires are consistent with those expected for each class.

2. A B KLFEDRVGTIK Endogenous peptide Synthetic peptide DANSFLQSV
Synthetic peptide B
DANSFLQSV
Endogenous peptide
Synthetic peptide

3. C GVYPMPGTQK Endogenous peptide Synthetic peptide
Synthetic peptide
Supplementary Figure 2: Tandem mass spectra of endogenous mutant peptides identified in 12T match to their corresponding synthetic peptides. (A) KLFEDRVGTIK, (B) DANSFLQSV and (C) GVYPMPGTQK.

4. KLFELNPLTGEWHY Endogenous peptide Synthetic peptide
Synthetic peptide
Supplementary Figure 3: Tandem mass spectra of endogenous mutant peptide identified in sample 55C match to the corresponding synthetic peptide.

5. LFIMISVWSF Endogenous peptide Synthetic peptide
Synthetic peptide
Supplementary Figure 4:Tandem mass spectra of endogenous mutant peptide identified in sample 86B match to the corresponding synthetic peptide.

6. A B
0.915
0.969
RNA expression 42B
RNA expression 51B
RNA expression 42A
RNA expression 51A C
0.872
RNA expression 55B
RNA expression 55A
Supplementary Figure 5: High similarity of gene expression between different tumor metastases. The log2 of the genes RNA expression (TPM values) were plotted. Pearson correlations are indicated in red. (A) patient 42, (B) patient 51 and (C) patient 55.

7. A B C D Patient 51 Patient 86 Patient 55 Patient 42
Class II
0.496
0.626
Class II
0.498
0.316
Class I
Class I
0.728
0.417
0.508
0.3
Intensity 51B Intensity 51A Intensity 51C
Intensity 86C Intensity 86B Intensity 86A
0.799
0.711
0.271
0.231
Intensity 51C Intensity 51A Intensity 51B
Intensity 86A Intensity 86B Intensity 86C C D
Patient 55
Patient 42
Class II
0.487
0.279
0.366
Class I
0.133
Class I
0.574
0.452
0.607
Intensity 42B
Class II
0.436
Intensity 55B Intensity 55C Intensity 55D Intensity 55A
0.317
0.436
0.402
Intensity 42A
0.36
0.591
0.217
Intensity 55A Intensity 55D Intensity 55C Intensity 55B
Supplementary Figure 6: Great similarity between the peptides derived from the different tumor metastases. The log2 of the peptides intensities were plotted for HLA-I and HLA-II peptides. Unique peptides from each sample were given a constant value of 15. Peptides derived from TAAs are marked in red. Pearson correlations are indicated in red for HLA-I and in blue for HLA-II. (A) patient 51, (B) patient 86, (C) patient 55 and (D) patient 42.

8. A
0.577
0.625
Frequency in 42B
Frequency in 51B
# Clones 1
2-10
11-100
>100
Frequency in 42A
Frequency in 51A
0.619
0.305
Frequency in 55B
Frequency in 86B
Frequency in 55A
Frequency in 86A B C 1
0.75
0.5
0.25 A B
Gini index value A A A B B A B A B
Patient
Supplementary Figure 7: TCR frequencies show high similarity of the most frequent TCRs between metastases from the same patient and different clonality levels of the metastases. (A) The frequencies of the different TCRs were plotted to compare the different metastases from the same patient. Since in the lower frequencies we can find many different TCRs with the same frequency in both metastases, we color coded the number of TCRs represented by each dot. Pearson correlation is indicated in red. (B) The productive frequency of the top-1, -10, -50 and -100 most frequent TCRs in the different tumor metastases. (C) Gini index value represents the clonality of each metastasis TCR population.

9. A
Frequency in 42A Frequency in 42B 3 2 2 3 B
Frequency in 55A Frequency in 55B 4 6 3 4 C
Frequency in 86A Frequency in 86B 3 4 4 4
Supplementary Figure 8: Most convergent TCR sequences were detected on both metastases from the same patient. The frequency of the nucleotide sequences of the two most convergent amino acid TCR sequences detected in the tumors TCR sequencing are shown in the bars. Each nucleotide sequence represented by layer, with the overlapping sequences presented by the same color in both metastases. (A) patient 42, (B) patient 55 and (C) patient 86.

10. A
T=0min
T=60min
Melanoma
12TILs B
Supplementary Figure 9: High specificity of TILs to their autologous melanoma in in vitro imaging analysis. (A) In vitro imaging of the killing of 12T melanoma cells by 12TILs. The arrows indicate the cells that had been lysed throughout the hour-long imaging session. Size bar is 50µm. (B) Survival of melanoma cells from in vitro imaging of three fields (T-test, p-value <0.0001).

11. A B
12T
12TILs
***
Only melanoma
Melanoma + TILs
TILs injection
Tumor volume (mm3)
Days after tumor injection C D
12T melanoma cells
12TILs
Blood vessels
12T melanoma 12TILs 108TILs E F
***
***
Track mean speed (μM/min)
Average minimal distance
from closest neighbor (μM)
108TIL 12TIL
108TIL 12TIL
Supplementary Figure 10: Intravital 2-photon microscopy of 12T tumors in NSG mice show specific killing by autologous TILs. (A) NSG mice were inoculated with 12T melanoma cells, and 7 days later treated, or not, with 12TILs. TILs significantly reduced tumor volume (significant interaction by repeated measure ANOVA followed by Bonferroni's post hoc test, p-value<0.001). Error bars show standard deviation of four replicates. In TFM expreiments (B-F) 12TILs were adoptively transferred to NGS mice bearing 12T tumors and were imaged by intravital 2-photon microscopy following I.V. injection of red Quantum dots. Size bar represents 50µm. (B) Membrane blabbing indicative of dying 12T melanoma cells can be observed (marked with arrows), (C) TILs concentrate in the vicinity of blood vessels (mean distance to the nearest blood vessel is 31um). Based on the density of blood vessels, two area could be observed. In the perivascular region (<50 µm from vessels) TIL density was 20,000 cells/mm3, and in the avascular area fewer TILs were observed  and TIL density was only ~6,000 cells/mm3. (D) Intravital 2-photon microscopy image of 12TILs and 108TILs that were injected to NSG mice bearing 12T tumor. Size bar is 50µm. (E) Track mean speed of 12TILs and 108TILs in the presence of 12T melanoma cells (Mann-Whitney test, p-value<0.0001). (F) Average minimal distance of TILs from its closest autologous TIL (Mann-Whitney test, p-value<0.0001).

12. Peptide concentration [μM]
IFNγ [pg/ml] B C
Peptide concentration [μM]
Supplementary Figure 11: The neo-antigens identified in 55C tumor and 12T melanoma cells are reactive compared to the wild-type peptide version. IFN-γ release measured after overnight co-culture of the TILs with EBV-transformed B cells that were pulsed with tittered concentrations of the mutated or wild-type peptides and express the relevant HLA allele. Error bars represent standard deviation of triplicates.

13. CUL4B L477F CTT>TTT
OLFML3 L355F CTC>TTC
SNRPA F38C GGC>TGC
MED15 P677S CCC>TCC
NCAPH2 S174Y TCC>TAC
TPD52L2 S123 TCG>TTG
Supplementary Figure 12: Mutated alleles of the identified neo-antigens in 12T are expressed in the melanoma cells. The area surrounding each of the mutations site was sequenced from the cells cDNA to examine if the mutated allele of the gene is expressed.

14. Supplementary Figure 13: The reactivity of the predicted neo-antigens of patient 51. IFN-γ release measured after overnight co-culture of the TILs with EBV transformed B cells that were pulsed with 10μM of the 25mer peptides.

15. Supplementary Figure 14: The TAAs identified in the tumor metastases are reactive. Antigenicity of the TAAs was determined by IFN-γ release after overnight co-culture of the TILs with EBV-transformed B cells that were pulsed with 10μM of the peptides. The intensity of the peptides is shown in the scale bar (X108). The TILs used for patient 51 were derived from metastasis 51A, for patient 92 from metastasis 92B and for patient 55 from metastasis 55D. Peptides that marked in grey were identified only in the HLA peptidomics of the 55D cell line (55D CL).

16. A B --- Frequency equality --- Threshold for statistical comparison1 3 4
Enriched in the 4-1BB positive sample
Enriched in the bulk sample
Not significant
Excluded from analysis
Frequency in bulk TIL sample
--- Frequency equality
--- Threshold for statistical comparison 11
Frequency in 4-1BB positive sample B
Clone number
sequence
Frequency in 4-1BB
Frequency in bulk
P-value 1
CATSDLGGPMNTEAFF
6.63E-02
5.95E-02
6.93E-61 11
CAISDYRTGGSSPLHF
1.00E-01
7.60E-05 3
CASSYNLGKPQHF
8.81E-06 4
CASREGPLNTEAFF
6.11E-02
4.02E-02
3.73E-53 7
CASSLRSSGGFTGELFF
2.34E-02
1.95E-02
1.83E-05 8
CASTSASPKQYF
8.48E-03
6.86E-03
Supplementary Figure 15: Enrichment of neo-antigens TCRs in the 4-1BB sample. (A) we used the immunoSEQ analyzer to identifies sequences that are differentially abundant between the bulk and 4-1BB positive samples. The sequences marked in blue dots were enriched in the 4-1BB positive sample. (B) The frequency of the clones which were enriched in the 4-1BB sample, p-value calculated by binomial two-sided test with Benjamini Hochberg correction. The sequences that marked in red are against NCAPH2 and the sequences marked in blue are against MED15.

17. A B
Supplementary Figure 16: Neo-antigen specific clone against OSBPL8 was found in different metastases of patient 55. (A) TILs that were isolated from sample 55D and undergone REP were used to isolate the OSBPL8 neo-antigen specific T-cells and TCR sequencing. One clone was identified and it is marked in red. The table show the 10 most frequent TCR sequences in the bulk REP TIL. (B) The 10 most frequent TCR sequences in the TCR sequencing derived from 55A tumor, and their frequency in tumor 55B. The neo-antigen specific clone was found to be the third most frequent in tumor 55A TCRs, although this mutation was not found in the tumor WES. This clone was also found in another metastases (B) but in lower frequency.

18. A

19. B
Supplementary Figure 17: The complete co-repertoire of tumor antigens and their corresponding TILs. (A) Use of both HLA peptidomics and our prediction algorithm pipeline enabled the characterization of the antigen repertoire presented by the tumor cells. We identified one neo-antigen that overlaped between both methods and unique neo-antigens for each one of the methods. HLA peptidomics can reveal both neo-antigens that elicit an immune response as well as non-immunogenic neo-antigens whereas the prediction pipeline can identify only antigens to which the patients’ TIL had prior response to, even if they are no longer presented by the tumor cells. HLA peptidomics also allows us to identify TAAs that could be used as immune targets in combination with the identified neo-antigens. We also observed that the reactivity of each neo-antigen-specific T cell clone is usually higher then the reactivity of the TAAs clones. (B) The TIL repertoire can be divided according to the reactive and non-reactive TILs. The majority of the reactive TILs are against the identified neo-antigens.